Breaking a Dogma: High-Throughput Live-Cell Imaging in Real-Time with Hoechst 33342

Area of Science:

• Cell Biology
• Microscopy
• Toxicology

Background:

• Automated live cell imaging (LCI) is crucial for studying substance effects in vitro.
• Traditional cell counting via phase-contrast imaging is limited to specific cell types.
• Hoechst 33342 nuclear staining is desirable for accurate cell counting but historically considered cytotoxic for LCI.

Purpose of the Study:

• To re-evaluate the dogma that Hoechst 33342 is unsuitable for live cell imaging due to cytotoxicity.
• To determine optimized Hoechst 33342 concentrations and exposure parameters for long-term LCI.
• To establish a reliable method for accurate cell counting during LCI.

Main Methods:

• Optimized exposure parameters using a 4× objective for live cell imaging.
• Evaluated minimum effective Hoechst 33342 concentrations for LCI at 30-min intervals over 5 days.
• Assessed cytotoxicity, cell viability, proliferation, and signaling pathway impacts of various Hoechst concentrations.

Main Results:

• A Hoechst 33342 concentration of 57 × 10^-9 m significantly inhibited proliferation and impaired cell viability.
• Hoechst 33342 concentrations between 7 × 10^-9 m and 28 × 10^-9 m were found to be non-cytotoxic.
• These safe concentrations did not impact cell viability, proliferation, or signaling pathways during LCI.

Conclusions:

• The historical dogma regarding Hoechst 33342 cytotoxicity in LCI is no longer justified with optimized parameters.
• Safe concentrations of Hoechst 33342 enable accurate, long-term cell counting in live cell imaging.
• This adapted method can be used with standard fluorescence microscopes to assess cytotoxicity or transduction efficiency repeatedly over time.